AUTHENTICATION OF MEDICINAL PLANTS TRADED AS HERBAL DRUGS BY USING SYSTEMATICS AND PHYTOCHEMICAL CHARACTERIZATION BY SOFIA RASHID Department of Plant Sciences Quaid-i-Azam University Islamabad Pakistan 2016 AUTHENTICATION OF MEDICINAL PLANTS TRADED AS HERBAL DRUGS BY USING SYSTEMATICS AND PHYTOCHEMICAL CHARACTERIZATION A Thesis Submitted to the Quaid-i-Azam University in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY In Plant Sciences (Plant Systematics and Biodiversity) By SOFIA RASHID Department of Plant Sciences Quaid-i-Azam University Islamabad Pakistan 2016 This humble effort is dedicated to My Loving Family And Respected Teachers Whose prayers and affections are the source of strength and sign of success for my future CONTENTS TITLES Page No CHAPTER: 1 INTRODUCTION 1.1 Medicinal Plants as a Source of Herbal Medicine 01 1.2 Medicinal Plants Diversity in Pakistan 02 1.3 Medicinal Plants Trade 03 1.4 Standardization of Herbal Medicines 06 1.5 Risk Assessment Approaches of Herbal Drugs 09 1.6 Authentication of Crude Herbal Drugs 12 1.7 Taxonomic Evaluation 13 1.8 Pharmacognostic Evaluation 13 1.9 Phytochemical Evaluation 14 1.10 Chromatographic Evaluation 15 1.11 Background Justification of the Present Project 15 1.12 Objectives 17 CHAPTER: 2 MATERIALS AND METHODS 2.1 Collection and Preservation of Plant Material 18 2.2 Taxonomic Evaluation 18 2.2.1 Morphological Description 18 2.2.2 Palynological Studies 20 2.2.3 Foliar Epidermal Anatomy (LM) 20 2.3 Pharmacognostic Evaluation 21 2.3.1 Organoleptic Evaluation 22 2.3.2 Fluorescence and Solubility Analysis 22 2.3.3 Physicochemical Evaluation 22 2.3.3.1 Determination of Moisture Content 23 2.3.3.2 Determination of Ash Values 23 2.4 Phytochemical Evaluation 24 2.5 High Pressure Liquid Chromatography (HPLC) 26 2.6 Antioxidant Activity 28 2.7 Documentation of Indigenous Knowledge of Medicinal Plants 29 2.8 Assessment of Herbal Markets 30 2.9 Annexure 1: Key to Morphological Study 31 2.10 Annexure 2: Key to Pollen Study (LM & SEM) 32 2.11 Annexure 3: Key to Foliar Epidermal Anatomy (LM) 33 2.12 Annexure 4: Questionnaire for Ethnobotanical Data Collection 34 2.13 Annexure 5: Questionnaire for Market Assessment of Herbal Drugs 35 CHAPTER: 3 RESULTS AND DISCUSSION 3.1 Authentication of Herbal Drug Tukhm-e-Kalonji (Nigella sativa L.) 40 3.2 Authentication of Herbal Drug Tukhm-e-balango (Lallemantia royleana Benth.) 59 3.3 Authentication of Herbal Drug Belladona ( Atropa acuminata Royle.) 78 3.4 Authentication of Herbal Drug Dhatura ( Datura stramonium L.) 96 3.5 Authentication of Herbal Drug Chiraita ( Swertia cordata G. Don) 114 3.6 Authentication of Herbal Drug Zafran ( Crocus sativus L.) 132 3.7 Authentication of Herbal Drug Resha khatmi ( Althaea officinalis L.) 150 3.8 Authentication of Herbal Drug (Picrorhiza kurroa Royle ex Benth.) 167 3.9 Conclusion 184 3.10 Recommendations 185 CHAPTER: 4 REFERENCES 186 LIST OF PLATES Plate # Title Page # Plate 1 & 2 Collection sites of problematic medicinal plants 36 Plate 3 & 4 Collection of medicinal plant in the field 36 Plate 5 & 6 Collection of traditional knowledge of medicinal plants 36 Plate 7 & 8 Observation of herbal drugs during market surveys 37 Plate 9 & 10 Data collection from herbalist and hakims 37 Plate 11 & 12 Light and Scanning electron microscopy (LM & SEM) 37 Plate 13 & 14 Powder sample for analysis and Desicator for constant weight 38 Plate 15 & 16 Determination of Moisture content and Ash analysis 38 Plate 17 & 18 HPLC and Spectrophotometer for phytochemical analysis 38 LIST OF TABLES Table # Title Page # Table 1 List of problematic medicinal plants of the study 19 Table 2 Authentication of herbal drug Tukhm-e-kalonji ( Nigella sativa L.) in 40 comparison with its adulterant Table 3 Comparative qualitative pollen morphology of Nigella sativa and Allium 43 cepa Table 4 Comparative quantitative pollen morphology of Nigella sativa and Allium 43 cepa Table 5 Comparative qualitative characters of foliar epidermal anatomy of Nigella 44 sativa and Allium cepa Table 6 Comparative quantitative characters of foliar epidermal anatomy of Nigella 44 sativa and Allium cepa Table 7 Fluorescence and solubility analysis of powdered drug of Nigella sativa 47 (Cold method) Table 8 Fluorescence and solubility analysis of powdered drug of Nigella sativa (Hot 48 method) Table 9 Fluorescence and solubility analysis of powdered drug of Allium cepa (Cold 49 method) Table 10 Fluorescence and solubility analysis of powdered drug of Allium cepa (Hot 50 method) Table 11 Physicochemical Parameters of Nigella sativa and Allium cepa 51 Table 12 Antioxidant activity of Ascorbic acid, Nigella sativa and Allium cepa 51 Table 13 Authentication of herbal drug Tukhm-e-balango ( Lallemantia royleana 59 Benth.) in comparison with its adulterant Table 14 Comparative qualitative pollen morphology of Lallemantia royleana and 62 Ocimum basilicum Table 15 Comparative quantitative pollen morphology of Lallemantia royleana and 62 Ocimum basilicum Table 16 Comparative qualitative characters of foliar epidermal anatomy of 63 Lallemantia royleana and Ocimum basilicum Table 17 Comparative quantitative characters of foliar epidermal anatomy of 63 Lallamentia royleana and Ocimum basilicum Table 18 Fluorescence and solubility analysis of powdered drug of Lallemantia 66 royleana (Cold method) Table 19 Fluorescence and solubility analysis of powdered drug of Lallemantia 67 royleana (Hot method) Table 20 Fluorescence and solubility analysis of powdered drug of Ocimum basilicum 68 (Cold method) Table 21 Fluorescence and solubility analysis of powdered drug of Ocimum basilicum 69 (Hot method) Table 22 Physicochemical Parameters of Lallemantia royleana and Ocimum basilicum 70 Table 23 Antioxidant activity of Ascorbic acid, Lallemantia royleana and Ocimum 70 basilicum Table 24 Authentication of herbal drug Belladona ( Atropa acuminata Royle.) in 78 comparison with its adulterant Table 25 Comparative qualitative pollen morphology of Atropa acuminata and 81 Solanum nigrum Table 26 Comparative quantitative pollen morphology of Atropa acuminata and 81 Solanum nigrum Table 27 Comparative qualitative characters of foliar epidermal anatomy of Atropa 82 acuminata and Solanum nigrum Table 28 Comparative quantitative characters of foliar epidermal anatomy of Atropa 82 acuminata and Solanum nigrum Table 29 Fluorescence and solubility analysis of powdered drug of Atropa acuminata 85 (Cold method) Table 30 Fluorescence and solubility analysis of powdered drug of Atropa acuminata 86 (Hot method) Table 31 Fluorescence and solubility analysis of powdered drug of Solanum nigrum 87 (Cold method) Table 32 Fluorescence and solubility analysis of powdered drug of Solanum nigrum 88 (Hot method) Table 33 Physicochemical Parameters of Atropa acuminata and Solanum nigrum 89 Table 34 Antioxidant activity of Ascorbic acid, Atropa acuminata and Solanum 89 nigrum Table 35 Authentication of herbal drug Dhatura ( Datura stramonium L.) in 96 comparison with its adulterant Table 36 Comparative qualitative pollen morphology of Datura stramonium and 99 Xanthium strumarium Table 37 Comparative quantitative pollen morphology of Datura stramonium and 99 Xanthium strumarium Table 38 Comparative qualitative characters of foliar epidermal anatomy of Datura 100 stramonium and Xanthium strumarium Table 39 Comparative quantitative characters of foliar epidermal anatomy of Datura 100 stramonium and Xanthium strumarium Table 40 Fluorescence and solubility analysis of powdered drug of Datura stramonium 103 (Cold method) Table 41 Fluorescence and solubility analysis of powdered drug of Datura stramonium 104 (Hot method) Table 42 Fluorescence and solubility analysis of powdered drug of Xanthium 105 strumarium (Cold method) Table 43 Fluorescence and solubility analysis of powdered drug of Xanthium 106 strumarium (Hot method) Table 44 Physicochemical Parameters of Datura stramonium and Xanthium 107 strumarium Table 45 Antioxidant activity of Ascorbic acid, Datura stramonium and Xanthium 107 strumarium Table 46 Authentication of herbal drug Chiraita ( Swertia cordata G. Don) and its 114 adulterant Table 47 Comparative qualitative pollen morphology of Swertia cordata and Swertia 117 paniculata Table 48 Comparative quantitative pollen morphology of Swertia cordata and Swertia 117 paniculata Table 49 Quantitative characters of foliar epidermal anatomy of Swertia cordata and 118 Swertia paniculata Table 50 Quantitative characters of foliar epidermal anatomy of Swertia cordata and 118 Swertia paniculata Table 51 Fluorescence and solubility analysis of powdered drug of Swertia cordata 121 (Cold method) Table 52 Fluorescence and solubility analysis of powdered drug of Swertia cordata 122 (Hot method) Table 53 Fluorescence and solubility analysis of powdered drug of Swertia paniculata 123 (Cold method) Table 54 Fluorescence and solubility analysis of powdered drug of Swertia paniculata 124 (Hot method) Table 55 Physicochemical Parameters of Swertia cordata and Swertia paniculata 125 Table 56 Antioxidant activity of Ascorbic acid, Swertia cordata and Swertia paniculata 125 Table 57 Authentication of herbal drug Zafran ( Crocus sativus L.) in comparison with 132 its adulterant Table 58 Comparative qualitative pollen morphology of Crocus sativus and 135 Carthamus tinctorius Table 59 Comparative quantitative pollen morphology of Crocus sativus and 135 Carthamus tinctorius Table 60 Comparative qualitative characters of foliar epidermal anatomy of Crocus 136 sativus and Carthamus tinctorius Table 61 Comparative